Thromboxane Receptors Antagonists

and/or Synthase Inhibitors

Giovanni Davı, Francesca Santilli, and Natale Vazzana

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

2 TP Receptors and Their Antagonism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

2.1 Molecular and Cellular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

2.2 TP Receptor Signaling in Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

2.3 TP Receptor Signaling in Vascular Endothelial

and Smooth Muscle Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

3 Drugs Affecting TXA2 Action: Other than COX Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

3.1 Inhibitors of Thromboxane Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

3.2 Dual TXS Inhibition/TP Antagonism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

4 Dual COXIB/TP Antagonists: A Possible New Twist in NSAID Pharmacology and

Cardiovascular Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

4.1 TP Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

5 Pathophysiological Rationale for the Superiority of TP-Receptor Antagonists Over

Aspirin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

5.1 Advantages as an Antithrombotic Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

5.2 Relevance of TP Receptors Inhibition in Atherosclerotic Disease . . . . . . . . . . . . . . . . . . 274

5.3 Ischemic Stroke: The Reasons of a Choice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

6 Great Expectations Disappointed: Did Terutroban

Fail to Perform, or PERFORM Did Fail? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

6.1 Preliminary Data Revisited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

6.2 The PERFORM Design Revisited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

7 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

8 Conclusions and Implications for Clinical Usefulness

of TP Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

G. Davı (*) • F. Santilli • N. Vazzana

Internal Medicine, University of Chieti, Chieti, Italy

e-mail: gdavi@unich.it

P. Gresele et al. (eds.), Antiplatelet Agents, Handbook of Experimental

Pharmacology 210, DOI 10.1007/978-3-642-29423-5_11,# Springer-Verlag Berlin Heidelberg 2012

261

Abstract Atherothrombosis is the major cause of mortality and morbidity in

Western countries. Several clinical conditions are characterized by increased inci-

dence of cardiovascular events and enhanced thromboxane (TX)-dependent platelet

activation. Enhanced TX generation may be explained by mechanisms relatively

insensitive to aspirin. More potent drugs possibly overcoming aspirin efficacy may

be desirable. Thromboxane synthase inhibitors (TXSI) and thromboxane receptor

antagonists (TXRA) have the potential to prove more effective than aspirin due to

their different mechanism of action along the pathway of TXA2. TXSI prevent the

conversion of PGH2 to TXA2, reducing TXA2 synthesis mainly in platelets, whereas

TXRA block the downstream consequences of TXA2 receptors (TP) activation.

TXA2 is a potent inducer of platelet activation through its interaction with TP on

platelets. TP are activated not only by TXA2, but also by prostaglandin (PG) D2,

PGE2, PGF2a, PGH2, PG endoperoxides (i.e., 20-HETE), and isoprostanes, all

representing aspirin-insensitive mechanisms of TP activation. Moreover, TP are

also expressed on several cell types such as macrophages or monocytes, and

vascular endothelial cells, and exert antiatherosclerotic, antivasoconstrictive, and

antithrombotic effects, depending on the cellular target.

Thus, targeting TP receptor, a common downstream pathway for both platelet

and extraplatelet TXA2 aswell as for endoperoxides and isoprostanes, may be a useful

antiatherosclerotic and a more powerful antithrombotic intervention in clinical

settings, such as diabetes mellitus, characterized by persistently enhanced

thromboxane (TX)-dependent platelet activation through isoprostane formation and

low-grade inflammation, leading to extraplatelet sources of TXA2. Among TXRA,

terutroban is an orally active drug in clinical development for use in secondary

prevention of thrombotic events in cardiovascular disease. Despite great expectations

on this drug supported by a large body of preclinical and clinical evidence and

pathophysiological rationale, the PERFORM trial failed to demonstrate the superior-

ity of terutroban over aspirin in secondary prevention of cerebrovascular and cardio-

vascular events among ~20,000 patients with stroke. However, the clinical setting and

the design of the study in which the drug has been challenged may explain, at least in

part, this unexpected finding.

Drugs with dual action, such as dual TXS inhibitors/TP antagonist and dual

COXIB/TP antagonists are currently in clinical development. The theoretical

rationale for their benefit and the ongoing clinical studies are herein discussed.

Keywords Antiplatelet therapy • Atherothrombosis • Ischemic stroke • Platelet

activation • Thromboxane biosynthesis • TP antagonists

1 Introduction

Atherosclerosis and its clinical manifestations (i.e., ischemic heart disease, cere-

brovascular or peripheral artery disease) are major causes of mortality and morbid-

ity in Western countries.

262 G. Davı et al.

Conventional antiplatelet agents such as aspirin or clopidogrel are currently used

in the prevention of cardiovascular events. However, more effective drugs with less

bleeding or gastrointestinal complications are desirable.

Thromboxane (TX) A2 is involved in a diverse range of physiological and

pathophysiological processes, including thrombosis, asthma, myocardial infarction

(MI), inflammation, acquired immunity, and atherogenesis. Thus, the stimulation of

TX/endoperoxide receptors (TP) elicits diverse biological effects under both nor-

mal and pathological conditions. Stimulation of TP results in activation of different

signaling cascades that regulate the cytoskeleton, cell adhesion, motility, nuclear

transcription factors, proliferation, cell survival, and apoptosis (Nakahata 2008).

TXA2 is the major product of the arachidonic acid (AA) metabolism in platelets

that, in response to various stimuli, is produced via the consequent actions of

cyclooxygenase (COX) and TX synthase (TXS). Through its interaction with TP

receptors on platelets, TXA2 is a potent inducer of platelet activation (Davı and

Patrono 2007).

Furthermore, the activation of endothelial TP promotes the expression of adhe-

sion molecules and favors adhesion and infiltration of monocytes/macrophages. TP

receptors exhibit a wide distribution within the cardiovascular system; in fact, these

receptors are membrane-bound G protein-coupled receptors (GPCR) found not only

on platelets but also on circulating inflammatory cells, such as macrophages or

monocytes, and in vascular endothelial cells, and smooth muscle cells (Meja et al.

1997; Miggin and Kinsella 1998).

Thus, antagonists of TP may have some advantages over aspirin as they not only

block the effect of TXA2 on platelets, but also inhibit other ligands such as

prostaglandin (PG) endoperoxides and isoprostanes. Because of the wide distribu-

tion of TP receptors in platelets, in the vascular wall or in atherosclerotic plaques,

TP antagonists inhibit also the effects of TXA2 over TP receptors on vascular cells

or in the plaque. Therefore, antagonists of TP receptors may not only have

antiplatelet effects but also impact endothelial dysfunction and the inflammatory

component of atherosclerosis (Chamorro 2009).

2 TP Receptors and Their Antagonism

2.1 Molecular and Cellular Biology

TP receptor is highly distributed in various tissues. In addition to platelets, endo-

thelial cells, smooth muscle cells, glomerular mesangial cells, cardiac myocytes,

and many other cells express the TP receptor (Fig. 1) (Nakahata 2008).

The multiple cellular signaling and regulatory mechanisms activated through TP

have been only recently characterized. In particular, it has been reported that

reactive oxidant species (ROS)-dependent upregulation of TP expression may

increase TXA2/isoprostane responses. This mechanism may significantly contribute

Thromboxane Receptors Antagonists and/or Synthase Inhibitors 263

to platelet activation and atherothrombosis in several clinical setting associated

with enhanced oxidative stress, such as hypercholesterolemia, obesity, and type

2 diabetes mellitus (T2DM). In addition, posttranscriptional modifications of the

receptor, such as phosphorylation or glycation, also determine TP internalization or

ligand desensitization. Whether a TP antagonist may affect this receptorial cross-

talk and these regulatory pathways is still unanswered.

TXA2 is the major product of the AA metabolism in platelets that, in response to

various stimuli, is produced via the consequent actions of COX and TXS (Davı and

Patrono 2007). Low-dose aspirin inhibits platelet TXA2 production through perma-

nent inactivation of the COX activity of the enzyme prostaglandin G/H-synthase

(Fig. 2), and it has clearly been shown to be cardioprotective in several clinical

settings (Patrono et al. 2005b). However, many nucleated cells and tissues produce

TXA2 in response to proinflammatory signals and oxidative stress, which acts in an

autocrine or paracrine manner as aspirin-insensitive TP agonists. TP receptors are

activated not only by TXA2, but also by PGD2, PGE2, PGF2a, PGH2, PG

endoperoxides (i.e., 20-HETE), and isoprostanes (Fig. 2).

By binding to TP receptor, these molecules activate several signaling cascades

which regulate endothelial cell activation (i.e., adhesion molecules expression),

vascular smooth muscle cell (VSMC) contraction, and platelet aggregation,

thereby accelerating progression of atherosclerotic lesions (Dogne et al. 2005)

(Fig. 3).

Platelets

Shape changeAggregationSecretion

Endothelial cells

Adhesion moleculesPGI2Angiogenesis

Smooth muscle cells

Vascular toneProliferationBronchoconstriction

Physiological Response

TP receptor

Kidney

Cell proliferationFibrosisGlomerulosclerosis

Cancer cells

AngiogenesisMetastatization

TXA2

Monocytes/Macrophages

Adhesion moleculesInfiltration

Fig. 1 TP receptor expression and functions. The thromboxane (TX) A2 receptor (TP) is

expressed in a variety of tissues and cells such as platelets, endothelial cells, vascular and

bronchial smooth muscle cells, kidney, monocyte/macrophages, and cancer cells. TP activation

in these cells is critically involved in the pathobiology of a wide range of diseases, including

atherothrombosis, hypertension, renal disease, immune/inflammatory disease, and cancerogenesis.

Abbreviations: PGI2 prostacyclin

264 G. Davı et al.

In addition to platelets and endothelial cells, TP receptors are also expressed in

other cell types involved in atherothrombosis, such as VSMCs (Miggin and

Kinsella 1998), macrophages, and monocytes (Meja et al. 1997) (Fig. 1). More

recently, it has been reported that TP is expressed in prostate cancer where it

regulates cell motility and cytoskeleton reorganization, thus representing a novel

target of antineoplastic interventions.

2.2 TP Receptor Signaling in Platelets

TXA2 positively amplify platelet aggregation and is responsible for platelet aggre-

gation in the presence of low concentrations of aggregatory stimuli, such as

epinephrine and thrombin. This effect is fully reversed by aspirin or selective

antibodies blocking TXS (Mehta et al. 1986).

PLA2

AA

PGH2 TXA2

TP

COX-1

TXS

COX-1 inhibitors(i.e. Low-dose aspirin)

TXS inhibitors

TP Antagonists

ROS

PGH2PGE220-HETE 8-iso-PGF2a

Dual-acting drugs

Platelet activation, Vasoconstriction,

Inflammatory response

ExtraplateletCOX-1

COX-2

Fig. 2 Acting sites of drugs affecting the TXA2/TP pathway. Low-dose aspirin is an irreversible

inhibitor of the cyclooxygenase (COX) activity of platelet prostaglandin (PG) H2 synthase-1.

Thromboxane synthase (TXS) inhibitors (i.e., ozagrel) block the conversion of PGH2 to TXA2.

However, TXS antagonism may increase PGH2, which acts on TP as an agonist, thereby

counteracting the antithrombotic efficacy. Dual-acting drugs (TXS inhibitors/TP antagonists:

ridogrel, picotamide) and TP antagonists (terutroban) act downstream by blocking the activation

of the thromboxane receptor (TP) by TXA2 as well as other prostanoids, including PGH2, PGE2,

endoperoxides, and F2-isoprostane. Abbreviations: HETE hydroxyeicosatetraenoic acid

Thromboxane Receptors Antagonists and/or Synthase Inhibitors 265

Both TXA2 and its precursor PGH2 bind the TP receptor in platelets. TXA2 is

more potent than PGH2 in initiating aggregation in platelet-rich plasma with EC50

of 66 � 15 nM and 2.5 � 1.3 mM, respectively. Conversely in washed platelets,

PGH2 is more potent than TXA2 with EC50 values of 45 � 2 and 163 � 21 nM,

respectively. However, whether PGH2 may significantly contribute to the responses

attributed to TXA2 in vivo is still to be investigated (Mayeux et al. 1988).

In washed human platelets, PGF2a and PGD2 display lower affinity interactions

to TP receptor (Mayeux et al. 1988). Individual prostanoid receptors display

~20–30% sequence identity and encode specific motifs common only to members

of the subfamily of PG receptors. Given their structural similarities, PGs may

activate more than one subtype of PG receptor. F2 isoprostanes are nonenzymatic,

free radical-catalyzed products of AA relatively more stable than TXA2. These

molecules, in particular 8-iso-PGF2a, have been shown to bind TP and modulate the

adhesive reactions and activation of platelets induced by low levels of other

agonists.

Inactive PLTs

Activated PLTs

PGH2, PGE2, TXA2

ROS

TP receptors

Atherothrombosis

NO NOPGI2

Dysfunctional Endothelium

PLTs Aggregation

Healthy Endothelium

Low-grade Inflammation/Oxidative Stress

COX-2

COX-1/2

8-iso-PGF2

TXA2

2, PGH2PGG2, PGH2

PGI2

Atherosclerosis

VSMCs

Fig. 3 The role of TXA2/TP receptor pathway in atherothrombosis. Within the atherosclerotic

plaque, the TP receptors are widely expressed on platelets (PLTs), endothelial cells, vascular

smooth muscle cells (VSMCs), circulating monocytes, and resident macrophages. Stimulation of

TP receptors by their ligands (TXA2, prostaglandins, endoperoxides, and F2-isoprostanes)

activates several signaling cascades which regulate endothelial cell activation (i.e., adhesion

molecules expression), VSMCs contraction, and platelet aggregation, thereby accelerating pro-

gression of atherosclerotic lesions. Abbreviation: NO nitric oxide

266 G. Davı et al.

2.3 TP Receptor Signaling in Vascular Endothelialand Smooth Muscle Cells

Increased vascular tone due to generation of prostanoids is a main feature of

endothelial dysfunction. In fact, endothelium dysfunction is characterized by an

increased production of prostanoids (i.e., TXA2), which facilitate the penetration of

macrophages in the vessel wall (Feletou et al. 2009). On endothelial cells, TXA2

activates the expression of adhesion proteins, such as ICAM-1, VCAM-1, and

endothelial leukocyte adhesion molecule-1 (ELAM-1) (Ishizuka et al. 1996). TP-

receptor dependent expression of ICAM-1, VCAM-1, and ELAM-1 is mediated by

protein kinase C (Ishizuka et al. 1998). TP activation also stimulates the expression

of leukocytes adhesion molecules (LAM) on endothelial cells (Ashton et al. 2003).

TP activation also promotes prostacyclin (PGI2) production from endothelial cells

through a negative feedback counterregulatory response (Cheng et al. 2002). In

fact, PGI2 attenuates platelet aggregation and VSMC contraction.

In several cardiovascular diseases, endothelial dysfunction is the result of the

release of endothelium-derived contracting factors (EDCF) that counteract the

vasodilator effect of nitric oxide (NO) produced by the endothelial NO synthase.

These endogenous TP agonists produced by the vascular endothelium cause

endothelium-dependent contraction and contribute to endothelial dysfunction, a

key factor in atherogenesis. Endothelium-dependent contractions involve activation

of COXs, production of ROS along with that of EDCFs, which diffuse toward the

vascular smooth muscle cells and activate their TP.

Besides the activity of endothelial COX-1, the activation of TP-receptors on

VSMCs is also relevant (Swinnen et al. 2009). TP-receptor activation stimulates

VSMC proliferation and hypertrophy (Uehara et al. 1988), by potentiating the mito-

genic effects of platelet derived growth factor (PDGF) and by increasing the synthesis

and release of endogenous basic fibroblast growth factor (bFGF) (Ali et al. 1993).

3 Drugs Affecting TXA2 Action: Other than COX Inhibitors

The dramatic success story of aspirin as antiplatelet drug had a paradoxically

negative effect on the development of drugs that work by closely related but distinct

mechanisms to that of aspirin (Fig. 2). TXS inhibitors and TP antagonists were

perceived as too close to aspirin to compete effectively with this inexpensive and

effective drug.

3.1 Inhibitors of Thromboxane Synthase

The inhibitors of TXS prevent the conversion of PGH2 to TXA2. These drugs

reduce TXA2 synthesis mainly in platelets and may improve TXA2-mediated

Thromboxane Receptors Antagonists and/or Synthase Inhibitors 267

pathophysiological conditions, such as thrombosis formation and thrombosis-related

disorders (Dogne et al. 2004). TXS inhibitors also enhance vascular generation of

PGI2, which prevents platelet aggregation induced by all known agonists (FitzGerald

et al. 1985). In fact, TXS inhibition leads to accumulation of PG endoperoxides in

platelets that may be donated to endothelial prostacyclin synthase at sites of platelet–-

vascular interactions (endoperoxide “steal”) (FitzGerald et al. 1985). Consistent with

this hypothesis, increased PGI2 generation in vivo has been reported after administra-

tion of several TXS inhibitors (FitzGerald et al. 1985). As PGI2 may inhibit platelet

activation by both TX-dependent and TX-independent mechanisms, it has been

proposed that TXS inhibitors may be more effective than aspirin to prevent

atherothrombosis.

Several TXS inhibitors—including dazoxiben, dazmagrel, pirmagrel, ozagrel,

isbogrel, and furegrelate—have been tested in clinical settings associated with

enhanced TX generation.

The greatest experience in human thrombotic disease has been gained with

dazoxiben. However, the benefits of this molecule in patients with coronary heart

disease were limited or absent (FitzGerald et al. 1985; Kiff et al. 1983; Thaulow

et al. 1984).

Ozagrel has been used clinically since 1992 in Japan for the treatment of asthma.

Treatment with ozagrel significantly reduced TX generation in patients with coro-

nary or cerebrovascular disease (Uyama et al. 1985; Yui et al. 1984). However, this

effect resulted into limited clinical benefit (Shikano et al. 1987).

It has been proposed that incomplete suppression of TX generation in vivo may

partly account for the lack of clinical efficacy of these drugs. In addition, TXS

inhibition increases PGI2 generation as well as the formation of other prostanoids,

including PGH2 and endoperoxides, which act as TP agonists, thereby

counteracting the reduction of TXA2-mediated events (Nakahata 2008).

3.2 Dual TXS Inhibition/TP Antagonism

Combined TXS inhibitors and TP antagonist may theoretically overcome the

limitations observed for TXS inhibitors. In fact, these drugs do not affect (or

enhance) PGI2 generation, while preventing TP activation by residual TX as well

as by other agonists (Patscheke 1990).

Accordingly, the combined administration of a dual TXS inhibitor/TP antagonist

gives stronger inhibition of platelet aggregation and prolongs bleeding time more

than either drug alone or acetylsalicylic acid (Patrono 1990).

Ridogrel is a drug developed more than 20 years ago as a more potent

antiplatelet agent than aspirin. Ridogrel is a TXA2 inhibitor with additional TP

antagonist properties that further enhance its antiaggregatory effects by diverting

endoperoxide intermediates into the PGI2 production pathway (Meadows and Bhatt

2007).

268 G. Davı et al.

Ridogrel has been studied primarily as an adjunctive agent to thrombolytic

therapy in acute MI. In 1993, animal studies showed that ridogrel limits MI size

after mechanical coronary occlusion and reperfusion at doses enhancing coronary

thrombolysis by streptokinase (Meadows and Bhatt 2007; Vandeplassche et al.

1993).

Thus, the Ridogrel Versus Aspirin Patency Trial (RAPT) was performed to

compare the efficacy and safety of ridogrel with that of aspirin as conjunctive

therapy for thrombolysis in patients with acute MI. However, despite positive

results from initial pilot studies, the largest clinical study, the RAPT, failed to

demonstrate any advantage with this agent over aspirin. In fact, in the study of 907

patients with acute MI, there was no difference in the primary end point of infarct

vessel patency rate between those randomized to ridogrel (72.2%) or aspirin

(75.5%). Despite ridogrel was not superior to aspirin in enhancing the fibrinolytic

efficacy of streptokinase, it was more effective in preventing new ischemic events

(The Ridogrel Versus Aspirin Patency Trial 1994).

In ulcerative colitis, local production of PGE2, PGI2, and TXA2 has been

demonstrated. The inflammatory infiltrate in ulcerative colitis consists of polymor-

phonuclear leukocytes, mononuclear leukocytes, and macrophages, all of which

release considerable amounts of TXA2. Although PGE2 may have protective effects

on intestinal mucosa, TXA2 appears to promote the development of chronic inflam-

matory lesions in the bowel. Thus, an imbalance between the synthesis of

cytoprotective prostaglandins, such as PGE2, and of the pro-inflammatory TXA2

may play a role in the development of chronic inflammation and mucosal damage in

patients with ulcerative colitis.

Treatment with selective inhibitors of TXA2 synthesis, including ridogrel,

reduced the release of TXA2, tissue damage, and the development of chronic

inflammatory lesions in the colon. A pilot clinical trial in patients with chronic

ulcerative colitis demonstrated that high-dose ridogrel (300 mg twice daily) signifi-

cantly reduced colonic mucosal TXA2 release, to 31% of basal levels, without

significantly reducing the levels of protective PGE2. However, two multicentre,

randomized, double-blind studies failed to find significant differences in the pri-

mary efficacy outcome measure among two different doses of ridogrel and placebo.

Thus, there was no clear indication in either trial of an effective dose of ridogrel in

the treatment of ulcerative colitis (Tytgat et al. 2002).

Various mechanisms are likely responsible for the results observed with

ridogrel in clinical trials, including potentially ineffective TP inhibition with the

concentrations of ridogrel used in human studies. As such, there currently are no

clinical indications for preferential use of ridogrel over aspirin.

Another drug with dual action (TXS inhibition/TP antagonism) is picotamide. In

a double blind, randomized trial (ADEP), 2,304 patients with intermittent claudi-

cation were allocated to receive picotamide or placebo. However, the trial showed

only a nonsignificant benefit of picotamide versus placebo in patients with periph-

eral artery disease (PAD) (Basili et al. 2010; Neri Serneri et al. 2004).

More recently, picotamide has been studied in diabetics with PAD randomized

to receive picotamide or aspirin for 2 years (DAVID study). Overall mortality, the

Thromboxane Receptors Antagonists and/or Synthase Inhibitors 269

predefined primary end point, was significantly lower among patients who received

picotamide (3.0%) than in those who received aspirin (5.5%) with a relative risk

ratio for picotamide versus aspirin of 0.55 (95% CI 0.31–0.98%). Conversely, the

combined end point of mortality and morbidity has a slightly lower incidence in the

picotamide group, but this difference does not reach statistical significance (Neri

Serneri et al. 2004). The results of this study should be cautiously interpreted in the

light of its limited statistical power and sample size. In fact, as confirmed by a

recent meta-analysis comparing the efficacy of different antiplatelet treatments in

patients with PAD, picotamide, like aspirin, is not associated with a statistically

significant reduction in cardiovascular adverse events (Violi and Hiatt 2007).

Conversely, a significant reduction in cardiovascular risk is observed with

thienopyridines, suggesting that the presence of PAD may render platelet activation

more critically dependent on ADP than on TXA2 release.

4 Dual COXIB/TP Antagonists: A Possible New Twist in NSAID

Pharmacology and Cardiovascular Risk

In the early 1990s, a new class of nonsteroidal anti-inflammatory drug (NSAID)

became available (COX-2 inhibitors, or COXIBs). The gastrointestinal safety,

dependent upon lack of COX-1 inhibition, coupled with the emerging evidence of

cardiovascular hazard associated with COXIBs, leading to withdrawal of rofecoxib

and valdecoxib, suggested that a potentially safer pharmacological approach

should be combining the anti-inflammatory activity of COXIBs together with a

cardioprotective component which might involve antagonism of TP receptors. This

could be achieved by making a simple combination of existing drugs targeted

against COX-2 or the TP receptor.

The possibility to combine powerful anti-inflammatory activity with TP antago-

nism within a single chemical entity provides the basis for a novel class of safer

NSAIDs and to plan highly innovative studies of structure–activity relationships,

chemical syntheses, and pharmacological investigations.

It has recently been demonstrated (Selg et al. 2007) that a traditional NSAID

(diclofenac) and a selective COXIB (lumiracoxib) possess an additional activity:

weak competitive antagonism at the TP receptor (Rovati et al. 2010). However, in

light of the importance of maintaining a fine balance between TP receptor antago-

nism activity and COX-2 inhibition, the co-administration of two different

molecules is not the best approach because it may result in significantly different

pharmacokinetic profiles. Combining both activities into a single chemical entity

represents a far better strategy (Selg et al. 2007): in fact, developing a compound

with a more “balanced” pharmacological profile relative to these two activities may

help evaluating if blocking the activity of TP might counterbalance the deleterious

cardiovascular effects driven by the PGI2 inhibition observed for COX-2 inhibitors.

270 G. Davı et al.

4.1 TP Antagonists

Several TP antagonists have been developed since the early 1980s. Development of

the earliest TP antagonists has been stopped because of their toxicity (or moderate

activity) in clinical situations, whereas others have not been investigated for

cardiovascular indication, and only the more recent TP antagonists reached clinical

evaluation for their antithrombotic properties (Dogne et al. 2004).

Thus, many TP antagonists have been developed for the treatment of TP-mediated

diseases, such as ifetroban, sulotroban, daltroban, linotroban, ramatoroban, and

seratrodast. Among them, seratorodast is an orally active TP antagonist used clinically

for the treatment of asthma in Japan since 1997 (Nakahata 2008).

However, evidence has been accumulated for a competitive TP antagonist,

terutroban (S-18886), as a potential candidate for atherothrombosis treatment, for

blocking atherosclerosis progression, and for transforming lesions towards a more

stable phenotype.

Terutroban is an orally active TP antagonist in clinical development for use in

secondary prevention of thrombotic events in cardiovascular disease. Terutroban has

been developed as a highly specific, high-affinity TP antagonist. Binding studies show

that the drug displaces the binding of [3H]-SQ29548 on human platelet membranes

with aKi value of 0.65 nmol/L, and theKdvalue for binding of [3H]-S18886 to human

platelet membranes averaged 0.96 nmol/L (Zuccollo et al. 2005). Unlike aspirin and

clopidogrel that bind their respective receptor in an irreversible manner, terutroban

has a reversible and dose-dependent antithrombotic effect within 96 h (Gaussem

et al. 2005).

Escalating doses of terutroban (30 and 100 mg/kg/day) compared with that of

aspirin (5 mg/kg/day) and clopidogrel (3 mg/kg/day) show a significant dose-

dependent effect on platelet aggregation. When used at higher doses, terutroban

is able to reduce collagen-dependent platelet aggregation, at least as well as

clopidogrel. Terutroban reduces platelet deposition on low shear (212 s�1) and

high shear (1,690 s�1) rate conditions (platelet thrombus formation in the Badimon

perfusion chamber) at least as well as clopidogrel, whereas aspirin does not have

any significant effect on platelet deposition. Thus, TP antagonists might be useful in

clinical settings characterized by severe arterial injury and high shear rate, such as

acute coronary syndromes and during and immediately after percutaneous

transluminal coronary angioplasty (Chamorro 2009).

Pharmacokinetics and pharmacodynamics of terutroban have been studied in

patients with PAD (Gaussem et al. 2005). Peak plasma levels are reached between

30 min and 2 h and the half life is 5.8–10 h. Maximal inhibition of U46619-induced

platelet aggregation is achieved within 1 h, and this effect is maintained for at least

12 h. Over the range of studied doses, there is a predictable relation between plasma

drug concentration and degree of platelet inhibition. Plasma concentrations above

10 ng/mL strongly inhibit U46619-induced platelet aggregation. These plasma

concentrations are reached only by dosages higher than 10 mg/day (Gaussem

et al. 2005).

Thromboxane Receptors Antagonists and/or Synthase Inhibitors 271

The antithrombotic effects of increasing doses (1–30 mg/day) of terutroban have

also been demonstrated in PAD (TAIPAD study) using a design based on the

ex vivo evaluation of platelet aggregation. This effect was predictable, dose-

dependent with maximal inhibition at 1 h, and lasted for approximately 48 h at

the oral dose of 30 mg (Fiessinger et al. 2010).

Terutroban does not bind other prostanoid receptors, such as IP or DP receptors,

and thus preserves antivasoconstrictive effects of their natural ligands, PGI2 and

PGD2 (Chamorro 2009).

5 Pathophysiological Rationale for the Superiority of

TP-Receptor Antagonists Over Aspirin

5.1 Advantages as an Antithrombotic Agent

An increased incidence of cardiovascular events and enhanced TX-dependent

platelet activation characterize several clinical conditions, and aspirin is thought

to be the best choice in these settings. However, enhanced TX generation may be

explained by several mechanisms relatively insensitive to aspirin.

Monocytes and macrophages are the largest source of TXA2 and are capable of

newly synthesizing TXA2 via their COX-2 pathway, which has a higher threshold

of inhibition by aspirin than platelet COX-1. Thus, extraplatelet, nucleate sources of

TXA2 biosynthesis, possibly triggered by inflammatory stimuli, are less affected

than platelet TXA2 production by the once-daily regimen of administration and by

the low dose administered, and may be an additional reason for the less than

expected response to aspirin.

Moreover, in clinical settings characterized by enhanced platelet generation,

younger reticulated platelets are increased, platelet COX-2 expression is up-

regulated, and a consistent TX production may be driven by this enzymatic pathway

relatively insensitive to aspirin (Guthikonda et al. 2007; Santilli et al. 2009).

Moreover, TP antagonists block all TP agonists; these include not only TXA2

but also endoperoxide (i.e., 20-HETE) and several isoprostanes, nonenzymatic

products of fatty acid oxidation formed under conditions of increased oxidative

stress (Davı and Falco 2005) and which are not inhibited by aspirin (Fig. 2). Aspirin

has no effect on isoprostanes and can actually increase endoperoxide (i.e., HETE)

production by COX (Meade et al. 1993). Recently, it has been reported that the

signaling mechanism of flow-induced constriction of human and rat cerebral

arteries involves enhanced production of ROS, COX activity, and is mediated by

20-HETE via TP receptors (Toth et al. 2011).

Oxidative stress is responsible for enhanced peroxidation of AA to form biolog-

ically active F2-isoprostanes, such as 8-iso-PGF2a, that is widely recognized as a

reliable marker of lipid peroxidation both in vitro and in vivo (Davı and Falco 2005;

Patrono et al. 2005a).

272 G. Davı et al.

Concentrations of 8-iso-PGF2a in the range of 1 nmol/L to 1 mmol/L induce a

dose-dependent increase in platelet shape change, calcium release from intracellu-

lar stores, and inositol phosphates (Patrono et al. 2005a). Moreover, 8-iso-PGF2acauses dose-dependent, irreversible platelet aggregation in the presence of low

concentrations of collagen, ADP, AA, and PGH2/TXA2 analogs that, when acting

alone, fail to aggregate platelets (Patrono et al. 2005a). These effects are prevented

by TP receptor antagonists and 8-iso-PGF2a may cross-desensitize biochemical and

functional responses to TX mimetics. These properties may be relevant to settings

where platelet activation and enhanced free-radical formation coincide, such as in

T2DM.

Patients with T2DM have a two- to fourfold increase in the risk of coronary

artery disease, and patients with T2DM but without previous MI carry the same

level of risk for subsequent acute coronary events as nondiabetic patients with

previous MI (Schramm et al. 2008). Diabetic patients exhibit platelet “hyper-

reactivity” both in vitro and in vivo (Ferroni et al. 2004), (Davı et al. 1990)

enhanced platelet regeneration (Watala et al. 2005), coupled with biochemical

evidence of persistently enhanced TX-dependent platelet activation (Ferroni et al.

2004). Urinary TXA2 metabolites, reflecting the whole biosynthesis of TXA2 in the

body by platelet and extraplatelet sources, are significantly higher in diabetes, with

the absolute postaspirin values of 11-dehydro-TXB2 in diabetics being comparable

to nonaspirinated controls (Davı et al. 1990; Ferroni et al. 2004) and such residual

TXA2 biosynthesis despite low-dose aspirin treatment has been shown to be

predictive of vascular events in high-risk settings, including diabetes (Eikelboom

et al. 2002). Thus, elevated urinary 11-dehydro-TXB2 levels identify patients who

are relatively resistant to aspirin and who may benefit from alternative antiplatelet

therapies or treatments that more effectively block in vivo TXA2 production or

activity.

Aspirin remains the cornerstone of antiplatelet prophylaxis, but appears to have

limited benefit in diabetes (Baigent et al. 2009). TP antagonists, blocking the

interaction of both aspirin-sensitive and aspirin-insensitive agonists should theoret-

ically provide more potent protection against the anticipated detrimental effects of

platelet activation (Fig. 4).

The hypothesis that hyperglycemia-induced oxidant stress in T2DM might

trigger enhanced generation of 8-iso-PGF2a and that this compound might, in

turn, contribute to platelet activation is supported by the finding that 8-iso-PGF2aformation is correlated with the rate of TXA2 biosynthesis in this setting (Davı et al.

1999) and that intensive antidiabetic treatment is associated with a reduction in both

urinary 8-iso-PGF2a and TX metabolites excretion rates (Patrono et al. 2005a).

Thus, both COX-2-derived TXA2 and F2-isoprostanes may act as aspirin-

insensitive agonists of the platelet TP receptor, accounting for the less than com-

plete inhibition of platelet aggregation in T2DM.

Another mechanism advocated in favor of TP antagonism, stands in its capacity

to leave COX-1 and COX-2 or any pathway of prostanoid synthesis unaltered, thus

preserving the cardioprotective eicosanoid PGI2, an important homeostatic mecha-

nism of endothelial thromboresistance triggered by platelet activation.

Thromboxane Receptors Antagonists and/or Synthase Inhibitors 273

In contrast, depression of COX-2-derived PGI2 by traditional NSAIDS or

COXIBs as well as by anti-inflammatory doses of aspirin removes a constraint on

platelet COX-1-derived TXA2 and other agonists that elevate blood pressure,

promotes atherogenesis, and augments the thrombotic response to plaque rupture.

5.2 Relevance of TP Receptors Inhibition in AtheroscleroticDisease

Treatment with a TP antagonist, but not treatment with aspirin, inhibits atherogen-

esis in apo-E deficient mice (Cayatte et al. 2000), strongly suggesting that TP

antagonists could be superior to aspirin in preventing atheroma. In New Zealand

TXA2

Platelet

COX-1Platelet

COX-1

Extraplateletsources

Platelet activation

COX-2

COX-2 Low-dose aspirin

ROS

8-iso-PGF2α

TP antagonist

ROS

Inflammatorysignals

Fig. 4 Possible advantages of TP antagonism over aspirin. Low-dose aspirin irreversibly blocks

TXA2 generation by platelet COX-1. However, in high-risk patients, accelerated platelet turnover

may increase platelet COX-1 recovery of TXA2 biosynthesis as well as COX-2-generated TXA2.

In addition, enhanced oxidative stress and chronic inflammation further stimulated TXA2 genera-

tion by extraplatelet COX-1 and COX-2. Finally, lipid peroxidation by reactive oxidant species

(ROS) is able to generate biologically active F2-isoprostane. These molecules, in particular 8-iso-

PGF2a, have been shown to bind TP and modulate the adhesive reactions and activation of platelets

induced by low levels of other agonists. By blocking TP activation by TXA2 and other aspirin-

insensitive agonists, TP antagonists should theoretically provide more potent protection against

the detrimental effects of platelet activation

274 G. Davı et al.

white rabbits, terutroban induces regression of atherosclerotic lesions of the aorta

detected by magnetic resonance imaging. The concomitant reduction in indexes of

inflammation into the lesions, such as macrophages, apoptotic cells, matrix

metalloproteinase-1, endothelin-1, suggests that selective inhibition of TP receptor

may shift toward a more stable plaque phenotype (Chamorro 2009). Moreover,

injury-induced vascular proliferation is enhanced in mice genetically deficient in

the PGI2 receptor and is reduced in mice lacking the TP receptor or treated with

terutroban. Lack of both prostanoid receptors abolishes postinjury restenosis

(Cheng et al. 2002).

Endogenous TP agonists are produced by the vascular endothelium, especially

under pathological conditions, causing endothelium-dependent contraction and

contributing to endothelial dysfunction. Therefore, TP antagonists may counter-

act endothelial dysfunction in diseases such as hypertension and diabetes (Feletou

et al. 2010). In apoE�/� mice with streptozotocin-induced diabetes, terutroban

reduces aortic atherosclerotic area, with improvement of endothelium-dependent

relaxations to acetylcholine (Santilli et al. 2011). Thus, TP antagonism may

attenuate inflammation and atherogenesis in experimental diabetes.

Administration of terutroban to coronary artery disease or to high-cardiovas-

cular-risk patients on top of aspirin treatment improved endothelial function

assessed by measuring flow-mediated dilatation (FMD) in the brachial artery

(Belhassen et al. 2003; Lesault et al. 2011). The beneficial effect of terutroban

on FMD was detectable after the first intake and persisted up to the end of the

2-week treatment period (Lesault et al. 2011). Thus, TP antagonists can inhibit

prostanoid-mediated vasoconstriction associated with aging and/or cardiovascu-

lar risk factors related to increased oxidative stress and consequent up-regulation

of COX-1 and/or induction of COX-2 (Giannarelli et al. 2010).

5.3 Ischemic Stroke: The Reasons of a Choice

The at least theoretical advantages on platelet inhibition and the actions far beyond

its antithrombotic effect, including the antiproliferative and antiatherogenic

properties, raised the hypothesis that TP-receptor antagonism could play a role in

the clinical prevention of ischemic stroke. Preclinical findings supported this

concept, indicating a greater beneficial effect of TP receptor inhibition over aspirin

in a rat model of ischemic stroke (Gelosa et al. 2010). In a double-blind, parallel

group study in patients with previous ischemic stroke and/or carotid stenosis,

terutroban showed an antithrombotic activity superior to aspirin and similar to

clopidogrel plus aspirin (Bal Dit Sollier et al. 2009). These encouraging data

were the basis for undertaking the Prevention of cerebrovascular and cardiovascu-lar Events of ischemic origin with teRutroban in patients with a history oF ischemicstrOke or tRansient ischaeMic attack (PERFORM) study (Bousser et al. 2009).

Thromboxane Receptors Antagonists and/or Synthase Inhibitors 275

This trial was designed to demonstrate the superiority of terutroban over

aspirin in secondary prevention of cerebrovascular and cardiovascular events

among ~20,000 patients with stroke. The trial (ISRCTN66157730) was recently

halted on the basis of an interim analysis failing to support the superiority hypothe-

sis, after 19,120 patients were randomly assigned, with a mean follow-up of 28·3

months (Bousser et al. 2011).

6 Great Expectations Disappointed: Did Terutroban

Fail to Perform, or PERFORM Did Fail?

The PERFORM was stopped prematurely for futility after 19,120 patients were

randomly assigned, with a mean follow-up of 28 months (Bousser et al. 2011). The

investigators recorded no difference between terutroban and aspirin in the compos-

ite vascular primary end point, or any of the secondary or tertiary end points.

However, the rate of minor bleeding was slightly increased with terutroban.

This apparent discrepancy versus the above-mentioned “great expectations”

around terutroban, supported by a large body of preclinical and clinical evidence

and pathophysiological rationale, draws attention and raises concerns about the

interpretation of the encouraging preclinical data, as well as about the design of the

clinical trial testing the superiority hypothesis of this drug versus aspirin.

6.1 Preliminary Data Revisited

The negative results of the PERFORM trial failed to come up to the expectations

based on the rationale and the preliminary data supporting the superiority of

terutroban over aspirin.

Both the anticipated superior antithrombotic effect and the peculiar antiatherogenic

and antivasoconstrictive properties of TP antagonism need to be reconsidered in

light of the clinical evidence.

Most of the data supporting a more potent antiplatelet effect of terutroban over

aspirin relied upon ex vivo measurements of platelet function, such as optical

aggregation to classical agonists and models of thrombus formation (Bal Dit Sollier

et al. 2009; Fiessinger et al. 2010).

The apparent gap between these premises and the findings of PERFORM

draws attention to the limitations of ex vivo measurements of platelet function in

the characterization of platelet activation and inhibition in vivo: less than

ideal intrasubject and intersubject variability, poor reproducibility on repeated

measurements, variability of the TX-independent component of the different aggre-

gation signals (Santilli et al. 2009).

276 G. Davı et al.

Moreover, measurements of platelet function ex vivo provide an index of

capacity that by no means reflects the extent of platelet activation and inhibition

in vivo. Mechanism-based biochemical measurements would provide a more faith-

ful estimation of the antiplatelet effect of aspirin.

As earlier mentioned, another war horse supporting the expected superiority of

terutroban over aspirin stands in the preservation of the ability of the vasculature to

synthesize the cardioprotective eicosanoid PGI2, an important homeostatic mecha-

nism of endothelial thromboresistance. However, the low-doses of aspirin (100 mg

daily) employed vs. terutroban in the PERFORM trial have been previously shown

to only marginally reduce systemic PGI2 biosynthesis in heart failure and ischemic

heart disease patients, counterbalanced by a profound reduction in TX biosynthesis

(Santilli et al. 2010), consistent with the relative COX-1 selectivity achieved by a

once-daily regimen of low-dose aspirin (Patrono et al. 2008) and the primary role of

COX-2 in PGI2 biosynthesis.

The antivasoconstrictive effect of terutroban, increasing blood flow through

enhanced endothelium-dependent vasodilation, had the theoretical potential to

affect the incidence of ischemic stroke in the PERFORM population, where tradi-

tional cardiovascular risk factors associated with endothelial dysfunction are highly

prevalent. However, it has to be acknowledged that, despite several reports (Santos-

Garcia et al. 2009) suggest an association, no data until now show conclusive

evidence of a direct relation between endothelium-dependent contractions and the

risk of ischemic stroke.

Similarly, the antiproliferative properties of terutroban, shown in a murine

model of vascular injury-induced proliferation of the carotid artery, did not

translate into a clinical benefit. This result might have been anticipated by the

failure to prevent postcoronary angioplasty restenosis by previously developed

TX-prostaglandin receptor antagonists, the CARPORT and the M-HEART (Savage

et al. 1995; Serruys et al. 1991). However, none of these two studies were free of

limitations, the first being an uncontrolled experience relying on a single measure-

ment of an angiographic end point, the second including no measure of the degree

of synthesis inhibition achieved by aspirin or of TP blockade by either antagonist.

Moreover, the M-HEART based the superiority of terutroban over aspirin on the

ability of terutroban to preserve the antiproliferative effect of PGI2 biosynthesis

during angiography, which was short lived and suppressed by the concurrent aspirin

treatment in all patients undergoing PCI (Pratico et al. 2000).

The antiatherogenic properties of TP antagonists were considered as a relevant

plus, stimulating the preferential recruitment of patients with an atherothrombotic

cerebral ischemic event. However, even in this subgroup, no benefit was recorded

for terutroban compared with aspirin in the PERFORM, possibly because their

atheromatous lesions were already well advanced. This assumption is supported by

a study performed in a murine model of atherogenesis, showing that TP antagonism

inhibits initiation and early development of atherosclerotic lesions in mice, but

failed to induce regression of established atherosclerotic disease (Egan et al. 2005).

Thus, the clinical use of TP antagonists would be expected to be useful in the earlier

stages of disease, rather than in reversing accumulated plaque burden in patients

with diffuse, established atherosclerosis.

Thromboxane Receptors Antagonists and/or Synthase Inhibitors 277

6.2 The PERFORM Design Revisited

A few lessons might be drawn by the comparison of PERFORM findings with

similar trials of other antiplatelets vs. aspirin. PERFORM is the second largest

secondary prevention trial of an antiplatelet drug undertaken so far in patients with

cerebral ischemic events. The largest study, PROFESS (Sacco et al. 2008), com-

pared aspirin plus extended-release dipyridamole and clopidogrel in 20,232 patients

followed up for a mean of 30 months. This trial showed similar rates of recurrent

stroke with aspirin plus extended-release dipyridamole and with clopidogrel. Fail-

ure to achieve the superiority goal in both trials raises concerns about the clinical

setting in which selective TP receptor blockade might confer an advantage over

low-dose aspirin. Ischemic stroke might be a difficult setting as compared to MI,

but no trial so far is available to confirm or reject this speculative hypothesis.

Furthermore, the comparative analysis versus previously performed trials in the

same setting unravels the likelihood that the event rate observed in the PERFORM

might be someway lower than expected, thus affecting the statistical power of the

study. Although recent trials (ESPRIT and CSPS 2) have shown similar rates of

strokes with aspirin to those seen in PERFORM (Halkes et al. 2006; Shinohara et al.

2010), the stroke rate with aspirin in PERFORM should have been much higher

because of the enrolment of several patients during the period of highest risk for

recurrence (less than 3 months since the qualifying event) and because 52% of the

qualifying strokes were due to the stroke subtype with the poorer prognosis,

atherothrombosis (compared with about 30% in ESPRIT and CSPS 2). Even in

the PROFESS trial, which, similar to the PERFORM, enrolled patients who had

experienced an ischemic stroke for less than 90 days, the event rates were remark-

ably close to PERFORM, but the proportion of atherothrombotic infarcts was

significantly higher (67% vs. 28%) in PERFORM.

The choice of the lag time after the qualifying event, similar to the PROFESS

trial, might also have affected the effectiveness of the drug: in fact, patients could

be enrolled after the index event in PERFORM much sooner than in other recent

antiplatelet trials. While aspirin has proven benefits when given early after a stroke

(Chen et al. 2000), the efficacy of this strategy for other antiplatelets has not been

definitively proven.

The choice of the dose (30 mg once daily) has been an additional issue advocated

to explain the drug failure to exert superior effects on vascular events than aspirin.

However, the slight excess in minor bleeding with this dose suggests that, even if

higher doses may be more appropriate on the efficacy side, this benefit could be

offset by more hemorrhagic episodes, which seem proportional to the number of

TX-prostaglandin receptors bound by the drug.

The duration of follow-up appears as another critical and still unanswered issue:

given that only 15% of patients were followed up to or beyond 3 years, a difference

in treatment effect might have emerged later, since additional potential longer term

effects of terutroban cannot be excluded.

278 G. Davı et al.

As previously shown for aspirin (Baigent et al. 2009), improving background

risk-factor control could have blunted the ability of terutroban to outperform

aspirin. Nowadays, many patients with a history of occlusive vascular events

would have their risks of recurrence reduced substantially by statins, other modern

drugs, and vascular procedures. If so, and if the other interventions approximately

halve the recurrence risk, then the absolute benefit of adding an antiplatelet

to these other methods might be only about half as great as that of giving an

antiplatelet alone.

Finally, the observation that patients with a history of ischemic stroke before the

qualifying event had a lower event rate with terutroban than with aspirin is worth of

further attention. Although this finding could be attributable to chance, it is plausi-

ble that most of these patients would have been receiving aspirin before their

PERFORM qualifying event, thus the switch to a different antiplatelet drug appears

as a more effective strategy than continuation of aspirin despite having experienced

an event while on aspirin. Lower than expected response to aspirin which translates

into clinical failure might be an expected and relevant phenomenon in particular

subgroups of patients, such as those with diabetes mellitus (representing 27% and

28% of each arm, respectively). Trials that randomly assign patients with a break-

through event while on aspirin to a newer antiplatelet drug or to a different dosing

regimen, rather than to the original aspirin dose, could provide insights into this

issue. Perhaps terutroban could be tested for this specific issue or in clinical settings

where a benefit is anticipated, such as diabetes mellitus. In the PERFORM, only

about one out of five of the patient population had diabetes, thus potentially

affecting the statistical power of the study to test the superiority hypothesis in

diabetes.

7 Future Perspectives

As earlier mentioned, failure of the PERFORM does not preclude the possibility

that TP receptor antagonist may be an effective tool in the prevention of vascular

events in other clinical settings, such as diabetes, where the pathophysiological

premises for a beneficial role look more sound. This might be worth of ad hoc trials,

although the PERFORM findings are likely to discourage any other drug company

from pursuing this drug target.

Moving from the cardiovascular setting, an increasing body of evidence

provides the rationale for a role of TXS and TP receptor signaling in carcinogenesis,

which may arise as a “rescue” setting for the clinical development of these drugs.

TXS signaling has been implicated in the development and progression of

cancer, by acting on a range of tumor cell survival pathways, such as tumor cell

proliferation, induction of apoptosis, invasion and metastasis, and tumor cell

angiogenesis (Cathcart et al. 2010).

Increased expression of TXS and TP-b isoform has been found in the tissues of

patients with bladder cancer. TP-b receptor overexpression in patients with bladder

Thromboxane Receptors Antagonists and/or Synthase Inhibitors 279

cancer is associated with a poorer prognosis (Moussa et al. 2008). Studies in cell

lines and mice have indicated a potential significant role of the TX signaling

pathway in the pathogenesis of human bladder cancer. Stimulation of TP receptors

is associated with a mitogenic response and in phosphorylation of several kinases.

TP receptor antagonists augment in vitro and in vivo responses to cisplatin, by

reducing cell proliferation in vitro, increasing the time of tumor onset, and reducing

the rate of tumor growth in vivo (Moussa et al. 2008). These studies raise the

possibility that the TP-b receptor could serve as a novel therapeutic target in

bladder cancer and its presence and/or overexpression could be used as a predictor

of prognosis and dictate therapy. Recently, increased tissue levels of the TP-breceptor in patients with bladder cancer have been found to be mirrored by

increased urinary levels of TXB2, the major metabolite of TXA2, suggesting that

patients with bladder cancer may be followed for progression or remission of their

disease by quantitation of these substances in their urine (Moussa et al. 2011).

TXS is also overexpressed in nonsmall cell lung cancer (NSCLC), particularly in

the adenocarcinoma subtype. Selective TXS inhibition prevents proliferation and

induces apoptosis (Cathcart et al. 2011).

Clearly, further studies are needed to delineate the role of TXS and TP-breceptors in cancer and to address the challenge of their pharmacological inhibition

through a clinical development program.

8 Conclusions and Implications for Clinical Usefulness

of TP Antagonists

Several clinical conditions are characterized by increased incidence of cardiovas-

cular events and enhanced TX-dependent platelet activation.

Aspirin is thought to be the best choice in these settings. However, the optimum

regimen to suppress TX formation remains undefined. In fact, enhanced TX gener-

ation may be explained by mechanisms relatively insensitive to aspirin.

Extraplatelet, nucleate sources of TXA2 biosynthesis, possibly triggered by

inflammatory stimuli, and F2-isoprostane formation, reflecting ongoing in vivo

oxidative stress, can activate platelets via the platelet TP receptor thus escaping

inhibition of aspirin (Davı et al. 1990).

Thus, the relevance of the TP receptor in the pathogenesis of vascular diseases,

particularly in diabetes, may be due to the fact that not only TXA2, but other

eicosanoids including HETEs and isoprostanes are produced to such an extent as

to activate the TP receptor. Aspirin has no effect on isoprostanes which are formed

nonenzymatically from AA, and aspirin can actually increase HETE production by

COX (Meade et al. 1993).

In clinical settings characterized by enhanced platelet generation, younger

reticulated platelets are increased, and platelet COX-2 expression is up-regulated

and a consistent TX production may be driven by this enzymatic pathway, rela-

tively insensitive to aspirin (Guthikonda et al. 2007; Santilli et al. 2009).

280 G. Davı et al.

An antithrombotic intervention blocking TP may be required, as a common

downstream pathway for both platelet and extraplatelet TXA2 as well as for

isoprostanes. Aspirin does not inhibit isoprostane formation. Moreover, intraplatelet

or extraplatelet TX generation may be only partly inhibited by aspirin under certain

pathological conditions, at least at the usual low doses given for cardiovascular

protection.

Moreover, a TP antagonist has actions far beyond its antithrombotic effect

exerted on platelets and can be attributed to direct effects on endothelial and smooth

muscle cells within the blood vessel wall. These include effects on vascular

adhesion molecules, NO synthase expression and function, oxidant production,

and accumulation of extracellular matrix and advanced glycation end-products.

Thus, TP antagonists may represent an ideal tool to improve our knowledge on

the pathophysiology of cardiovascular diseases and to improve our pharmacologi-

cal “weapons” to counteract them in clinical settings, such as diabetes mellitus,

characterized by persistent enhanced TXA2-dependent platelet activation.

Knowledge Gaps

• The prevention of vascular events by TP receptor antagonists in clinical

settings, such as diabetes, where the pathophysiological premises for a

beneficial role look more sound needs to be defined.

• The hypothesis that TP-b receptor could serve as a novel therapeutic target

in bladder cancer and its presence and/or overexpression could be used as a

predictor of prognosis and dictate therapy needs to be tested.

• More in general, further studies are needed to delineate the role of TXS

and TP-b receptors in cancer and to address the challenge of their phar-

macological inhibition through a clinical development program.

Key Messages

• Thromboxane synthase (TXS) inhibitors and thromboxane receptor (TP)

antagonists have the potential to prove more effective than aspirin due to

their different mechanism of action along the pathway of TXA2. TXS

inhibitors prevent the conversion of PGH2 to TXA2, reducing TXA2

synthesis mainly in platelets, whereas TP antagonists block the down-

stream consequences of TP activation.

• Targeting TP receptor, a common downstream pathway for both platelet and

extraplatelet TXA2 as well as for endoperoxides and isoprostanes, may be a

useful antiatherosclerotic and a more powerful antithrombotic intervention

in clinical settings, such as diabetes mellitus, characterized by persistently

enhanced TX-dependent platelet activation through isoprostane formation

and low-grade inflammation, leading to extraplatelet sources of TXA2.(continued)

Thromboxane Receptors Antagonists and/or Synthase Inhibitors 281

• Despite great expectations on this drug supported by a large body of

preclinical and clinical evidence and pathophysiological rationale, the

PERFORM trial failed to demonstrate the superiority of terutroban over

aspirin in secondary prevention of cerebrovascular and cardiovascular

events among ~20,000 patients with stroke. However, the clinical setting

and the design of the study in which the drug has been challenged, as well

as a sometimes uncritical translation of preclinical data into a rationale for

a clinical trial, may explain, at least in part, this unexpected finding.

• Drugs with dual action, such as dual TXS inhibitors/TP antagonist and

dual COXIB/TP antagonists are currently in clinical development. Besides

the theoretical rationale for their benefit, ongoing clinical studies are

challenging their potential.

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